The goals of Project 4 are to analyze how the TIM gene alleles influence the confornnation and ligand binding properties ofthe TIM proteins, to uncover new ligand binding motif by structure deternnination and to understand how those motifs are engaged in ligand recognition. Our recent structure determination of murine TIM-1, TIM-2 and TIM-4 proteins have provided relevant insight on ligand binding by the TIM proteins. The structures identified a novel TIM-TIM interaction and a critical ligand binding cavity with a metal ion (metal ion-dependent ligand binding site, MILIBS). Ligands such as phosphatidylserine (PtdSer) bind into this unique pocket in TIM-1 and TIM-4 proteins. Recently we found that the TIM-3 protein binds also to PtdSer and that the binding affinity is TIM-3 allele dependent. We will study the influence ofthe mouse and human TIM-1 polymorphism on PtdSer recognition. The effects on cell survival or activation mediated by intracellular signals triggered by the binding of PtdSer to TIM proteins on the cell surface, and the differences among alleles will be analyzed together with other projects of the Program using different experimental models. We will determine the relevance ofthe MILIBS in ligand recognition. We will identify antibodies recognizing the MILIBS and blocking TIM protein functions. They will be used in other program projects for functional studies in animal models. We will characterize the involvement of this site in HAV binding and in homotypic TIM-TIM interactions. We will determine whether PtdSer containing vesicles can mediate TIMTIM interactions. We observed that the MILIBS was engaged on the accumulation ofthe mTIM-1 protein inside ofthe cell. We will extend the analysis of TIM protein trafficking to other TIM proteins and identify the nature ofthe intracellular vesicles containing TIM proteins. We will carry out structural studies on TIM proteins and ligands, and biophysical studies on TIM protein binding to PtdSer in cellular membranes. The structures will uncover ligand and antibody binding footprints and identify new motifs engaged on ligand recognition. We will carry out biophysical studies to uncover penetration ofthe MILIBS into a lipid bilayer upon TIM protein binding to membrane anchored PtdSer. Using structural techniques and mAbs we will identify conformations of the TIM proteins on the cell surface and its relation to domain composition, alleles and cell environment. The differential display of a TIM protein to ligands on the cell surface might be of relevance for modulating TIM protein functions.